VIRUSES
crossm Complete Genome Sequence of Escherichia coli Phage vB_EcoM_Alf5 Laurynas Alijošius, Eugenijus Šimoliu nas, Laura Kaliniene, Rolandas Meškys, Lidija Truncaite˙ Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania
ABSTRACT Here, we announce the complete genome sequence of the Escherichia coli myophage vB_EcoM_Alf5 belonging to the genus Felixo1virus, whose members have not been comprehensively studied at the molecular level. Phage vB_EcoM_Alf5 infects E. coli K-12-derived laboratory strains and therefore is well suited for functional studies.
P
athogenic Escherichia coli and Salmonella strains are the major causative agents of foodborne diseases in both humans and animals. With the emergence of multidrug-resistant strains, bacteriophages have been proposed as an alternative antimicrobial (1–7). A number of bacteriophages potentially suitable for the biocontrol of E. coli and Salmonella spp. have been isolated and sequenced, but only a few of them have been functionally characterized at the molecular level. In this study, we describe the complete genome sequence of the E. coli-specific myophage vB_EcoM_Alf5 (Alf5) capable of infecting E. coli K-12-derived laboratory strains, including KEIO collection mutants (8). Bacteriophage Alf5 was isolated from water samples collected from the Nemunas River in Lithuania using E. coli MH1 strain as a host. Transmission electron micrographs of phage particles showed that Alf5 virion morphology was indistinguishable from that of other Felixo1virus phages (9–11). The isolated genomic DNA of Alf5 was sequenced at BaseClear group in the Netherlands using Illumina HiSeq 2500 DNA sequencing technology. De novo assembly of the genome sequence data was carried out using CLC Genomics Workbench software version 8.5.1. The assembled contigs were then scaffolded using the SSPACE Premium scaffolder version 2.3, which resulted in a single scaffold of 87,662 bp in length (2,975-fold coverage), with a G⫹C content of 39.02%. The NCBI BLASTn tool was used to perform the whole-genome sequence alignments and subsequent phylogenetic analysis. The open reading frames (ORFs) in the Alf5 genome were predicted with Glimmer version 2.02 (12) and Geneious version 5.5.6 (13) and annotated based on the results of BLASTp analysis. tRNAscan-SE 1.21 (14) was used to identify tRNAs. BLASTn analysis revealed that the genome sequence of Alf5 is most similar to that of felixo1virus vB_EcoM-VpaE1 (VpaE1) (93% coverage, 96% identity), which infects E. coli B strains with an incomplete core lipopolysaccharide (11). The restriction digestion analysis in silico using Restriction Mapper version 3 tool (http://www .restrictionmapper.org) showed that the Alf5 genome sequence contains no recognition sites for many restriction enzymes, including BamHI, XhoI, SacI, PstI, and even MboI, which recognizes GATC, a trait also observed in other phages of the same genus (11). The genome of Alf5 has a total of 133 ORFs and 19 tRNAs. A large number of encoded tRNAs and homing endonucleases (3 in Alf5), as well as the presence of genes for RIIA, RIIB, lysin, and glutaredoxin, is a characteristic feature of Felixo1virus phages. With the exception of 2 putative tail fiber proteins (gp73 and gp74), the predicted Alf5 Volume 5 Issue 20 e00315-17
Received 14 March 2017 Accepted 28 March 2017 Published 18 May 2017 Citation Alijošius L, Šimoliu nas E, Kaliniene L, Meškys R, Truncaite˙ L. 2017. Complete genome sequence of Escherichia coli phage vB_EcoM_Alf5. Genome Announc 5:e00315-17. https://doi.org/10.1128/genomeA.00315-17. Copyright © 2017 Alijošius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Lidija Truncaite˙, [email protected].
genomea.asm.org 1
Alijošius et al.
structural proteins, as well as those involved in metabolism of nucleic acids, are most closely related to their counterparts found in bacteriophages VpaE1 and Felix 01 (86 to 100% identity). The most obvious differences between the genomes of Alf5 and other Felixo1virus phages are observed in the ORFs, which encode hypothetical and tail fiber proteins, possibly accounting for their host range. In conclusion, bacteriophage Alf5 is a good model for both analysis of the differences in the host ranges and functional characterization of genetically related Felixo1virus phages. Accession number(s). The complete genome sequence of vB_EcoM_Alf5 was deposited in GenBank under the accession number KX377933. ACKNOWLEDGMENT This work was supported by a grant (no. MIP-002/2014) from the Research Council of Lithuania.
REFERENCES 1. Niu YD, Johnson RP, Xu Y, McAllister TA, Sharma R, Louie M, Stanford K. 2009. Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7. J Appl Microbiol. 107:646 – 656. https://doi.org/10.1111/j .1365-2672.2009.04231.x. 2. Bardina C, Spricigo DA, Cortés P, Llagostera M. 2012. Significance of the bacteriophage treatment schedule in reducing Salmonella colonization of poultry. Appl Environ Microbiol 78:6600 – 6607. https://doi.org/10 .1128/AEM.01257-12. 3. Bardina C, Colom J, Spricigo DA, Otero J, Sánchez-Osuna M, Cortés P, Llagostera M. 2016. Genomics of three new bacteriophages useful in the biocontrol of Salmonella. Front Microbiol. 7:545. https://doi.org/10.3389/ fmicb.2016.00545. 4. Spricigo DA, Bardina C, Cortés P, Llagostera M. 2013. Use of a bacteriophage cocktail to control Salmonella in food and the food industry. Int J Food Microbiol 165:169 –174. https://doi.org/10.1016/j.ijfoodmicro .2013.05.009. 5. Kim M, Kim S, Park B, Ryu S. 2014. Core lipopolysaccharide-specific phage SSU5 as an auxiliary component of a phage cocktail for Salmonella biocontrol. Appl Environ Microbiol 80:1026 –1034. https://doi.org/ 10.1128/AEM.03494-13. 6. Bao H, Zhang P, Zhang H, Zhou Y, Zhang L, Wang R. 2015. Bio-control of Salmonella Enteritidis in foods using bacteriophages. Viruses 7:4836 – 4853. https://doi.org/10.3390/v7082847. 7. Colom J, Cano-Sarabia M, Otero J, Cortés P, Maspoch D, Llagostera M. 2015. Liposome-encapsulated bacteriophages for enhanced oral phage therapy against Salmonella spp. Appl Environ Microbiol 81:4841– 4849. https://doi.org/10.1128/AEM.00812-15. 8. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA,
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10.
11.
12.
13.
14.
Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008. https://doi.org/10.1038/msb4100050. Villegas A, She YM, Kropinski AM, Lingohr EJ, Mazzocco A, Ojha S, Waddell TE, Ackermann HW, Moyles DM, Ahmed R, Johnson RP. 2009. The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1. Virol J 6:41. https://doi.org/10.1186/1743-422X-6-41. Whichard JM, Weigt LA, Borris DJ, Li LL, Zhang Q, Kapur V, Pierson FW, Lingohr EJ, She YM, Kropinski AM, Sriranganathan N. 2010. Complete genomic sequence of bacteriophage Felix O1. Viruses 2:710 –730. https://doi.org/10.3390/v2030710. Šimoliu nas E, Vilkaityte˙ M, Kaliniene L, Zajancˇkauskaite˙ A, Kaupinis A, Staniulis J, Valius M, Meškys R, Truncaite˙ L. 2015. Incomplete LPS corespecific felix01-like virus vB_EcoM_VpaE1. Viruses 7:6163– 6181. https:// doi.org/10.3390/v7122932. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27: 4636 – 4641. https://doi.org/10.1093/nar/27.23.4636. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/ bioinformatics/bts199. Schattner P, Brooks AN, Lowe TM. 2005. The tRNAscan-SE, snoscan and snoGPS Web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33:W686 –W689. https://doi.org/10.1093/nar/gki366.
genomea.asm.org 2
crossm Complete Genome Sequence of Escherichia coli Phage vB_EcoM_Alf5 Laurynas Alijošius, Eugenijus Šimoliu nas, Laura Kaliniene, Rolandas Meškys, Lidija Truncaite˙ Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Life Sciences Center, Vilnius University, Vilnius, Lithuania
ABSTRACT Here, we announce the complete genome sequence of the Escherichia coli myophage vB_EcoM_Alf5 belonging to the genus Felixo1virus, whose members have not been comprehensively studied at the molecular level. Phage vB_EcoM_Alf5 infects E. coli K-12-derived laboratory strains and therefore is well suited for functional studies.
P
athogenic Escherichia coli and Salmonella strains are the major causative agents of foodborne diseases in both humans and animals. With the emergence of multidrug-resistant strains, bacteriophages have been proposed as an alternative antimicrobial (1–7). A number of bacteriophages potentially suitable for the biocontrol of E. coli and Salmonella spp. have been isolated and sequenced, but only a few of them have been functionally characterized at the molecular level. In this study, we describe the complete genome sequence of the E. coli-specific myophage vB_EcoM_Alf5 (Alf5) capable of infecting E. coli K-12-derived laboratory strains, including KEIO collection mutants (8). Bacteriophage Alf5 was isolated from water samples collected from the Nemunas River in Lithuania using E. coli MH1 strain as a host. Transmission electron micrographs of phage particles showed that Alf5 virion morphology was indistinguishable from that of other Felixo1virus phages (9–11). The isolated genomic DNA of Alf5 was sequenced at BaseClear group in the Netherlands using Illumina HiSeq 2500 DNA sequencing technology. De novo assembly of the genome sequence data was carried out using CLC Genomics Workbench software version 8.5.1. The assembled contigs were then scaffolded using the SSPACE Premium scaffolder version 2.3, which resulted in a single scaffold of 87,662 bp in length (2,975-fold coverage), with a G⫹C content of 39.02%. The NCBI BLASTn tool was used to perform the whole-genome sequence alignments and subsequent phylogenetic analysis. The open reading frames (ORFs) in the Alf5 genome were predicted with Glimmer version 2.02 (12) and Geneious version 5.5.6 (13) and annotated based on the results of BLASTp analysis. tRNAscan-SE 1.21 (14) was used to identify tRNAs. BLASTn analysis revealed that the genome sequence of Alf5 is most similar to that of felixo1virus vB_EcoM-VpaE1 (VpaE1) (93% coverage, 96% identity), which infects E. coli B strains with an incomplete core lipopolysaccharide (11). The restriction digestion analysis in silico using Restriction Mapper version 3 tool (http://www .restrictionmapper.org) showed that the Alf5 genome sequence contains no recognition sites for many restriction enzymes, including BamHI, XhoI, SacI, PstI, and even MboI, which recognizes GATC, a trait also observed in other phages of the same genus (11). The genome of Alf5 has a total of 133 ORFs and 19 tRNAs. A large number of encoded tRNAs and homing endonucleases (3 in Alf5), as well as the presence of genes for RIIA, RIIB, lysin, and glutaredoxin, is a characteristic feature of Felixo1virus phages. With the exception of 2 putative tail fiber proteins (gp73 and gp74), the predicted Alf5 Volume 5 Issue 20 e00315-17
Received 14 March 2017 Accepted 28 March 2017 Published 18 May 2017 Citation Alijošius L, Šimoliu nas E, Kaliniene L, Meškys R, Truncaite˙ L. 2017. Complete genome sequence of Escherichia coli phage vB_EcoM_Alf5. Genome Announc 5:e00315-17. https://doi.org/10.1128/genomeA.00315-17. Copyright © 2017 Alijošius et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Lidija Truncaite˙, [email protected].
genomea.asm.org 1
Alijošius et al.
structural proteins, as well as those involved in metabolism of nucleic acids, are most closely related to their counterparts found in bacteriophages VpaE1 and Felix 01 (86 to 100% identity). The most obvious differences between the genomes of Alf5 and other Felixo1virus phages are observed in the ORFs, which encode hypothetical and tail fiber proteins, possibly accounting for their host range. In conclusion, bacteriophage Alf5 is a good model for both analysis of the differences in the host ranges and functional characterization of genetically related Felixo1virus phages. Accession number(s). The complete genome sequence of vB_EcoM_Alf5 was deposited in GenBank under the accession number KX377933. ACKNOWLEDGMENT This work was supported by a grant (no. MIP-002/2014) from the Research Council of Lithuania.
REFERENCES 1. Niu YD, Johnson RP, Xu Y, McAllister TA, Sharma R, Louie M, Stanford K. 2009. Host range and lytic capability of four bacteriophages against bovine and clinical human isolates of Shiga toxin-producing Escherichia coli O157:H7. J Appl Microbiol. 107:646 – 656. https://doi.org/10.1111/j .1365-2672.2009.04231.x. 2. Bardina C, Spricigo DA, Cortés P, Llagostera M. 2012. Significance of the bacteriophage treatment schedule in reducing Salmonella colonization of poultry. Appl Environ Microbiol 78:6600 – 6607. https://doi.org/10 .1128/AEM.01257-12. 3. Bardina C, Colom J, Spricigo DA, Otero J, Sánchez-Osuna M, Cortés P, Llagostera M. 2016. Genomics of three new bacteriophages useful in the biocontrol of Salmonella. Front Microbiol. 7:545. https://doi.org/10.3389/ fmicb.2016.00545. 4. Spricigo DA, Bardina C, Cortés P, Llagostera M. 2013. Use of a bacteriophage cocktail to control Salmonella in food and the food industry. Int J Food Microbiol 165:169 –174. https://doi.org/10.1016/j.ijfoodmicro .2013.05.009. 5. Kim M, Kim S, Park B, Ryu S. 2014. Core lipopolysaccharide-specific phage SSU5 as an auxiliary component of a phage cocktail for Salmonella biocontrol. Appl Environ Microbiol 80:1026 –1034. https://doi.org/ 10.1128/AEM.03494-13. 6. Bao H, Zhang P, Zhang H, Zhou Y, Zhang L, Wang R. 2015. Bio-control of Salmonella Enteritidis in foods using bacteriophages. Viruses 7:4836 – 4853. https://doi.org/10.3390/v7082847. 7. Colom J, Cano-Sarabia M, Otero J, Cortés P, Maspoch D, Llagostera M. 2015. Liposome-encapsulated bacteriophages for enhanced oral phage therapy against Salmonella spp. Appl Environ Microbiol 81:4841– 4849. https://doi.org/10.1128/AEM.00812-15. 8. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA,
Volume 5 Issue 20 e00315-17
9.
10.
11.
12.
13.
14.
Tomita M, Wanner BL, Mori H. 2006. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:2006.0008. https://doi.org/10.1038/msb4100050. Villegas A, She YM, Kropinski AM, Lingohr EJ, Mazzocco A, Ojha S, Waddell TE, Ackermann HW, Moyles DM, Ahmed R, Johnson RP. 2009. The genome and proteome of a virulent Escherichia coli O157:H7 bacteriophage closely resembling Salmonella phage Felix O1. Virol J 6:41. https://doi.org/10.1186/1743-422X-6-41. Whichard JM, Weigt LA, Borris DJ, Li LL, Zhang Q, Kapur V, Pierson FW, Lingohr EJ, She YM, Kropinski AM, Sriranganathan N. 2010. Complete genomic sequence of bacteriophage Felix O1. Viruses 2:710 –730. https://doi.org/10.3390/v2030710. Šimoliu nas E, Vilkaityte˙ M, Kaliniene L, Zajancˇkauskaite˙ A, Kaupinis A, Staniulis J, Valius M, Meškys R, Truncaite˙ L. 2015. Incomplete LPS corespecific felix01-like virus vB_EcoM_VpaE1. Viruses 7:6163– 6181. https:// doi.org/10.3390/v7122932. Delcher AL, Harmon D, Kasif S, White O, Salzberg SL. 1999. Improved microbial gene identification with GLIMMER. Nucleic Acids Res 27: 4636 – 4641. https://doi.org/10.1093/nar/27.23.4636. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A. 2012. Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649. https://doi.org/10.1093/ bioinformatics/bts199. Schattner P, Brooks AN, Lowe TM. 2005. The tRNAscan-SE, snoscan and snoGPS Web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33:W686 –W689. https://doi.org/10.1093/nar/gki366.
genomea.asm.org 2
